Oxytocin improves treatment of obstructive sleep apnea

ABSTRACT

The present disclosure provides methods for treating obstructive sleep apnea (OSA) and OSA induced cardiorespiratory diseases. The disclosure provides, inter alia, methods for treating or alleviating: OSA or OSA induced hypertension, cardiac arrhythmias, myocardial ischemia, sudden cardiac death or stroke, by administering oxytocin. The disclosure further provides methods for improving sleep satisfaction in OSA patients by administering oxytocin.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/184,091, filed Nov. 8, 2018 (U.S. Pat. No. 10,842,845, issuedate of Nov. 24, 2020), which is a continuation of U.S. patentapplication Ser. No. 15/327,252, filed Jan. 18, 2017 (now U.S. Pat. No.10,166,268, issued Jan. 1, 2019), which is a 371 national phase entry ofPCT Application No. PCT/US2015/038970, filed Jul. 2, 2015, which claimsthe benefit of priority to U.S. Provisional Application No. 62/028,972,filed on Jul. 25, 2014, the contents of each of which are herebyincorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This disclosure was made with Government support under contractR01-HL72006 awarded by the NIH. The U.S. Government has certain rightsin the disclosure.

FIELD OF THE DISCLOSURE

The present disclosure relates to methods and compositions for treatingobstructive sleep apnea (OSA) and OSA induced cardiorespiratorydiseases. More particularly, the disclosure relates to compositions andmethods that are useful for treating OSA and OSA induced hypertensionand cardiac dysfunction and to compositions and methods that are usefulfor improving sleep quality in OSA patients.

BACKGROUND

Patients with obstructive sleep apnea (OSA) experience repetitivecollapses of the upper airway during sleep causing intermittent periodsof hypoxia and hypercapnia (H/H) accompanied by arterial oxygendesaturations and increases in arterial carbon dioxide levels,ultimately altering both cardiac parasympathetic and sympathetic nervoussystem activity (Bradley and Floras, 2009; Leung, 2009; Loke et al.,2012). Upon termination of apneas, asphyxia causes a brief arousal fromsleep, sympathetic activity increases and vagal tone decreases leadingto surges in blood pressure (BP) and heart rate (HR) (Bradley andFloras, 2009; Leung, 2009; Loke et al., 2012). These acute effects ofOSA are thought to cause chronic long term changes in cardiovasculardysfunction including hypertension, arrhythmias, and cardiovascularmortality (Bradley and Floras, 2009). Indeed, patients suffering fromOSA have increases in blood pressure, lower heart rate variability, andreduced baroreflex sensitivity (Carlson et al., 1996; Trimer et al.,2013; Konecny et al., 2014), with chronic impairment in cardiacautonomic function i.e., sympathetic hyperactivity and diminishedparasympathetic activity (Trimer et al. 2013). While identification ofthe mechanisms underlying the elevations in sympathetic nerve activityin CIH and OSA has been the focus of numerous studies (Fletcher et al.,1999; Fletcher et al., 2002; Kc et al., 2010; Zoccal et al., 2011);studies identifying the characteristics and mechanisms underlyingdepressed cardiac parasympathetic activity are scarce.

Exposure to chronic intermittent hypoxia (CIH) or hypoxia/hypercapnia(CIH/H) during the sleeping period of animals mimics the repetitiveepisodes of H/H that occur in humans with OSA and thus, serve as ananimal model of OSA. Similar to what is observed in patients with OSA,animals exposed to CIH or CIH/H experience decreased baroreflexsensitivity, increased sympathetic activity, diminished parasympatheticactivity to the heart, and develop hypertension within 3 weeks of CIH/H(Carlson et al, 1996; Dyavanapalli et al., 2014; Lai et al., 1985;Parish and Somers, 2004; Pinol et al., 2014).

The parasympathetic activity to heart arises from cardiac vagal neurons(CVNs) located in the nucleus ambiguus (NA) and dorsal motor nucleus ofthe vagus (DMNX) that dominate the control of heart rate (Mendelowitz1999) (FIG. 1). The preganglionic vagal efferent nerve terminals of theCVNs synapse with the postganglionic intracardiac ganglia neuronslocated within the connective and fat tissue surrounding sinoatrial andatrioventricular nodes (Armour 2008). CVNs are typically intrinsicallysilent and thus depend on synaptic inputs (glutamatergic, GABAergic, andglycinergic) to dictate their activity (Mendelowitz 1996; Willis et al.1996; Neff et al. 1998; Wang et al. 2001; Wang et al. 2003).

The paraventricular nucleus of the hypothalamus (PVN) is critical incontrolling autonomic function under normal conditions and regulatingcardiovascular activity in response to hypoxic stress. The adversealterations in BP, HR, and respiration that occur with CIH have beenpostulated to involve pathways from the PVN to sympathetic brainstemnuclei. Recently, it has been hypothesized that different PVN neuronsprojecting to parasympathetic nuclei, particularly the dorsal vagalcomplex (DVC) where parasympathetic cardiac control originates,differentially alter autonomic balance (Kc and Dick, 2010). However,much less is known concerning the function and role of theneurotransmission from the PVN to parasympathetic areas of the brainstemin normal and disease states. Consequently, there is a great need in themedical community for understanding the mechanisms underlying theparasympathetic control of cardiac dysfunction and for the developmentof novel therapeutic compounds, compositions, and methods of treatment,which help alleviate the aforementioned cardiorespiratory side effectsassociated with OSA.

The present disclosure investigates the mechanisms responsible fordiminished parasympathetic control of cardiac functions during OSA andshows that oxytocin-secreting PVN neurons, as well as administration ofoxytocin, are novel and powerful targets to mitigate important negativecharacteristics of the apnea as well as the adverse cardiorespiratoryconsequences of OSA.

SUMMARY OF THE DISCLOSURE

The present disclosure provides methods of treatment and compositionsfor treating or alleviating obstructive sleep apnea (OSA) and OSAinduced cardiorespiratory diseases and compromised sleep quality.

In aspects, the present disclosure provides methods for treating oralleviating OSA, and OSA induced compromised sleep quality andcardiorespiratory diseases such as hypertension, cardiac arrhythmias,myocardial ischemia, sudden cardiac death, and stroke, said methodscomprising, inter alia, administering an effective dose of oxytocin.

In aspects, the disclosure provides that many of the events associatedwith OSA such as duration of apnea, arousals per hour, increased bloodpressure, increased heart rate, oxygen desaturation and compromisedsleep quality can be reduced or inhibited by administration of oxytocin.

In some aspects, the present disclosure provides for an improved methodof OSA treatment, which comprises administering an effective dose ofoxytocin in conjunction with continuous positive airway pressure (CPAP)therapy. In one embodiment, the effective dose of oxytocin may beadministered to OSA patients receiving CPAP therapy via the CPAPinhalation system.

In one embodiment, the disclosure provides a method for treating apatient suffering from obstructive sleep apnea, comprising:administering to the patient an effective dose of oxytocin.

In one embodiment, the disclosure provides a method for treating apatient suffering from OSA induced cardiorespiratory disease,comprising: administering to the patient an effective dose of oxytocin.Cardiorespiratory diseases that may be treated or alleviated from themethods of the present disclosure are selected from the group consistingof: hypertension, cardiac arrhythmias, myocardial ischemia, suddencardiac death, and stroke. In another embodiment, the disclosureprovides a method for treating a patient suffering from OSA inducedhypertension, comprising: administering to the patient an effective doseof oxytocin.

In some embodiments, the disclosure provides a method for improvingsleep quality in a patient suffering from obstructive sleep apnea,comprising: administering to the patient an effective dose of oxytocin.

In one embodiment, the patient suffering from obstructive sleep apnea isreceiving CPAP therapy.

In one embodiment, the effective dose of oxytocin is administeredintranasally. In one aspect, the effective dose of oxytocin is about 20International Units (IU) per day. In another aspect, the effective doseof oxytocin is about 30 IU per day. In yet another aspect, the effectivedose of oxytocin is about 40 IU per day. In still another aspect, theeffective dose of oxytocin is at least 40 IU per day.

In one embodiment, oxytocin is administered closer to the sleeping timeof the patient. In another embodiment, oxytocin is administered about 10minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or about 1 hourprior to sleeping. In yet another embodiment, oxytocin is administeredwithin an hour of the patient falling asleep.

In one aspect, the administration of oxytocin prevents or reduces therisk of developing OSA induced cardiorespiratory diseases in thepatient. In another aspect, the administration of oxytocin reduces thehypertension experienced by the patient. In yet another aspect, theadministration of oxytocin maintains or decreases the heart rate and/orblood pressure of the patient.

In one aspect, the administration of oxytocin improves sleep quality orsleep satisfaction in the patient. In another aspect, the administrationof oxytocin decreases the number of arousals per hour experienced by thepatient during sleep. In one aspect, the administration of oxytocindecreases the number of arousals per hour experienced by the patient byat least 10%. In still another aspect, the administration of oxytocinleads to a decrease in the duration of apnea experienced by the patientduring sleep. In one aspect, the administration of oxytocin decreasesthe duration of apnea experienced by the patient by at least 10%. Instill another aspect, the administration of oxytocin leads to a decreasein the oxygen desaturation experienced by the patient during sleep.

In some embodiments, the disclosure provides a method for treating apatient suffering from obstructive sleep apnea induced hypertension,comprising: intranasally administering to the patient at least 40International Units (IU) of oxytocin within an hour of the patientfalling asleep.

In one embodiment, the disclosure provides a method for improving sleepquality in a patient suffering from obstructive sleep apnea, comprising:intranasally administering to the patient at least 40 InternationalUnits (IU) of oxytocin within an hour of the patient falling asleep,wherein the administration of oxytocin decreases the number of arousalsper hour experienced by the subject during sleep. In one aspect, theadministration of oxytocin leads to improvement in empirical factorsindicative of sleep quality in the patient.

In other embodiments, the disclosure provides a method for treating apatient suffering from obstructive sleep apnea induced hypertension andcompromised sleep quality, comprising: intranasally administering to thepatient at least 40 International Units (IU) of oxytocin within an hourof the patient falling asleep.

In still other embodiments, the disclosure provides a method fortreating obstructive sleep apnea in a patient receiving continuouspositive airway pressure (CPAP) therapy, comprising: administering tothe patient at least 40 International Units (IU) of oxytocinintranasally via the CPAP inhalation system within an hour of thepatient falling asleep.

In one aspect, the disclosure provides a method for treating obstructivesleep apnea in a patient receiving continuous positive airway pressure(CPAP) therapy, comprising: intranasally administering to the patientabout 5 International Units (IU) of oxytocin per hour via the CPAPinhalation system for about 8 hours. In another aspect, oxytocin isadministered to the patient intranasally via the CPAP inhalation systemat the rate of about 5.7 IU/hour for about 7 hours. In yet anotheraspect, oxytocin is administered to the patient intranasally via theCPAP inhalation system at the rate of about 6.6 IU/hour for about 6hours. In yet another aspect, oxytocin is administered to the patientintranasally via the CPAP inhalation system at the rate of about 8IU/hour over the sleep period of 5 hours.

In certain embodiments, the disclosure provides a method for treating apatient suffering from obstructive sleep apnea induced cardiorespiratorydisease, comprising: intranasally administering to the patient at least40 International Units (IU) of oxytocin within an hour of the patientfalling asleep.

In some embodiments, the disclosure provides a method for treating apatient suffering from obstructive sleep apnea, comprising: activatingoxytocin-secreting neurons in the paraventricular nucleus (PVN) of thehypothalamus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the schematic for parasympathetic control of cardiacfunction.

FIG. 2A shows changes in systolic and diastolic blood pressure from day1 (control) to day 28 of CIH/H exposure. Systolic (solid line) anddiastolic (dash line) blood pressures significantly increased from day 6and day 16, respectively, compared to day 1 control and reached tohypertensive levels by day 21. n=6; *p<0.05; One way ANOVA. The valuesrepresent an average blood pressure value recorded for 20 minutes duringexposure to air in days prior to and during CIH/H exposures. FIG. 2Bshows changes in heart rate in response to normoxia and an acute bout ofH/H (3 mins) at the onset and after 4 weeks of CIH/H exposure. n=6;*p<0.05; Student's paired t-test. FIG. 2C shows changes in mean arterialblood pressure (MAP) in response to normoxia and an acute bout of H/H (3mins) at the onset and after 4 weeks of CIH/H exposure. n=6; **p<0.05;Student's paired t-test.

FIG. 3A shows representative traces in control conditions showingGABAergic spontaneous IPSCs recorded from CVNs in the NA of unexposedanimals while applying glycinergic and glutamatergic blockers. FIG. 3Bshows representative traces in control conditions showing GABAergicspontaneous IPSCs recorded from CVNs in the NA of CIH/H exposed animalswhile applying glycinergic and glutamatergic blockers. FIG. 3C shows thequantitative histograms depicting the frequency of GABAergic IPSCs inCVNs in the NA of unexposed and CIH/H exposed animals. FIG. 3D showsrepresentative traces in control conditions showing GABAergicspontaneous IPSCs recorded from CVNs in the DMNX of unexposed animalswhile applying glycinergic and glutamatergic blockers. FIG. 3E showsrepresentative traces in control conditions showing GABAergicspontaneous IPSCs recorded from CVNs in the DMNX of CIH/H exposedanimals while applying glycinergic and glutamatergic blockers. FIG. 3Fshows the quantitative histograms depicting the frequency of GABAergicIPSCs in CVNs in the DMNX of unexposed and CIH/H exposed animals. Thenumbers in parentheses represent ‘n’ value. *p<0.05, unpaired t-test.

FIG. 4A shows representative traces in control conditions showingglycinergic IPSCs recorded from CVNs in the NA of unexposed animalswhile applying GABAergic and glutamatergic blockers. FIG. 4B showsrepresentative traces in control conditions showing glycinergic IPSCsrecorded from CVNs in the NA of CIH/H exposed animals while applyingGABAergic and glutamatergic blockers. FIG. 4C shows the quantitativehistograms depicting the frequency of glycinergic IPSCs in CVNs in theNA of unexposed and CIH/H exposed animals. FIG. 4D shows representativetraces in control conditions showing glycinergic IPSCs recorded fromCVNs in the DMNX of unexposed animals while applying GABAergic andglutamatergic blockers. FIG. 4E shows representative traces in controlconditions showing glycinergic IPSCs recorded from CVNs in the DMNX ofCIH/H exposed animals while applying GABAergic and glutamatergicblockers. FIG. 4F shows the quantitative histograms depicting thefrequency of glycinergic IPSCs in CVNs in the DMNX of unexposed andCIH/H exposed animals. The numbers in parentheses represent ‘n’ value.*p<0.05, unpaired t-test.

FIG. 5A shows representative traces in control conditions showingglutamatergic EPSCs recorded from CVNs in the NA of unexposed animalswhile applying GABAergic and glycinergic blockers. FIG. 5B showsrepresentative traces in control conditions showing glutamatergic EPSCsrecorded from CVNs in the NA of CIH/H exposed animals while applyingGABAergic and glycinergic blockers. FIG. 5C shows the bar graphdepicting the frequency of glutamatergic EPSCs in CVNs in the NA ofunexposed and CIH/H exposed animals. FIG. 5D shows the bar graphdepicting the amplitude of glutamatergic EPSCs in CVNs in the NA ofunexposed and CIH/H exposed animals. FIG. 5E shows representative tracesin control conditions showing glutamatergic EPSCs recorded from CVNs inthe DMNX of unexposed animals while applying GABAergic and glycinergicblockers. FIG. 5F shows representative traces in control conditionsshowing glutamatergic EPSCs recorded from CVNs in the DMNX of CIH/Hexposed animals while applying GABAergic and glycinergic blockers. FIG.5G shows the bar graph depicting the frequency of glutamatergic EPSCs inCVNs in the DMNX of unexposed and CIH/H exposed animals. FIG. 5H showsthe bar graph depicting the amplitude of glutamatergic EPSCs in CVNs inthe DMNX of unexposed and CIH/H exposed animals. The numbers inparentheses represent ‘n’ value. *p<0.05, unpaired t-test.

FIG. 6A shows representative traces of GABAergic IPSCs recorded fromCVNs in the NA of unexposed animals in control conditions and followingH/H exposure for 10 minutes. FIG. 6B shows the histograms depicting thefrequency of GABAergic IPSCs recorded from CVNs in the NA of unexposedanimals in control conditions and H/H (10 minutes) conditions. FIG. 6Cshows the histograms depicting the amplitude of GABAergic IPSCs recordedfrom CVNs in the NA of unexposed animals in control conditions and H/H(10 minutes) conditions. FIG. 6D shows representative traces ofGABAergic IPSCs recorded from CVNs in the DMNX of unexposed animals incontrol conditions and following H/H exposure for 10 minutes. FIG. 6Eshows the histograms depicting the frequency of GABAergic IPSCs recordedfrom CVNs in the DMNX of unexposed animals in control conditions and H/H(10 minutes) conditions. FIG. 6F shows the histograms depicting theamplitude of GABAergic IPSCs recorded from CVNs in the DMNX of unexposedanimals in control conditions and H/H (10 minutes) conditions. FIG. 6Gshows representative traces of GABAergic IPSCs recorded from CVNs in theNA of CIH/H exposed animals in control conditions and following H/Hexposure for 10 minutes. FIG. 6H shows the histograms depicting thefrequency of GABAergic IPSCs recorded from CVNs in the NA of CIH/Hexposed animals in control conditions and H/H (10 minutes) conditions.FIG. 6I shows the histograms depicting the amplitude of GABAergic IPSCsrecorded from CVNs in the NA of CIH/H exposed animals in controlconditions and H/H (10 minutes) conditions. FIG. 6J shows representativetraces of GABAergic IPSCs recorded from CVNs in the DMNX of CIH/Hexposed animals in control conditions and following H/H exposure for 10minutes. FIG. 6K shows the histograms depicting the frequency ofGABAergic IPSCs recorded from CVNs in the DMNX of CIH/H exposed animalsin control conditions and H/H (10 minutes) conditions. FIG. 6L shows thehistograms depicting the amplitude of GABAergic IPSCs recorded from CVNsin the DMNX of CIH/H exposed animals in control conditions and H/H (10minutes) conditions.

FIG. 7A shows representative traces of glycinergic IPSCs recorded fromCVNs in the NA of unexposed animals in control conditions and followingH/H exposure for 10 minutes. FIG. 7B shows the histograms depicting thefrequency of glycinergic IPSCs recorded from CVNs in the NA of unexposedanimals in control conditions and H/H (10 minutes) conditions. FIG. 7Cshows the histograms depicting the amplitude of glycinergic IPSCsrecorded from CVNs in the NA of unexposed animals in control conditionsand H/H (10 minutes) conditions. FIG. 7D shows representative traces ofglycinergic IPSCs recorded from CVNs in the DMNX of unexposed animals incontrol conditions and following H/H exposure for 10 minutes. FIG. 7Eshows the histograms depicting the frequency of glycinergic IPSCsrecorded from CVNs in the DMNX of unexposed animals in controlconditions and H/H (10 minutes) conditions. FIG. 7F shows the histogramsdepicting the amplitude of glycinergic IPSCs recorded from CVNs in theDMNX of unexposed animals in control conditions and H/H (10 minutes)conditions. FIG. 7G shows representative traces of glycinergic IPSCsrecorded from CVNs in the NA of CIH/H exposed animals in controlconditions and following H/H exposure for 10 minutes. FIG. 7H shows thehistograms depicting the frequency of glycinergic IPSCs recorded fromCVNs in the NA of CIH/H exposed animals in control conditions and H/H(10 minutes) conditions. FIG. 7I shows the histograms depicting theamplitude of glycinergic IPSCs recorded from CVNs in the NA of CIH/Hexposed animals in control conditions and H/H (10 minutes) conditions.FIG. 7J shows representative traces of glycinergic IPSCs recorded fromCVNs in the DMNX of CIH/H exposed animals in control conditions andfollowing H/H exposure for 10 minutes. FIG. 7K shows the histogramsdepicting the frequency of glycinergic IPSCs recorded from CVNs in theDMNX of CIH/H exposed animals in control conditions and H/H (10 minutes)conditions. FIG. 7L shows the histograms depicting the amplitude ofglycinergic IPSCs recorded from CVNs in the DMNX of CIH/H exposedanimals in control conditions and H/H (10 minutes) conditions.

FIG. 8A shows representative action potential firing recorded incurrent-clamp configuration from a PVN OXT neuron expressing DREADDsbefore CNO and post CNO indicating excitatory DREADDs activation withCNO application significantly increases the firing of PVN OXT neurons.FIG. 8B shows quantitative bar charts depict the frequency of actionpotentials before and after CNO application in 7 PVN OXT neuronsexpressing DREADDs. *p<0.0001; one-way ANOVA.

FIG. 9A shows representative responses from CHO cells due to the releaseof oxytocin, as measured by activating oxytocin receptors and subsequentincrease in intracellular calcium levels, deposited onto brainstemtissue taken from animals chronically exposed to air, CIH/H, and CIH/Hwith daily PVN OXT neuron activation. FIG. 9B shows quantitative barcharts depicting the percent control fluorescence of CHO cell responses,as measured by an increase in intracellular calcium levels, in brainstemtissue taken from animals exposed to air (n=14), CIH/H (n=16), and CIH/Hwith daily OXT neuron activation n=17). *p<0.05; one-way ANOVA withrepeated measure. ⁺p<0.05; one-way ANOVA.

FIG. 10A shows changes in resting MAP before and after CNO injection toactivate PVN oxytocin neurons. MAP in oxytocin neuron activated animalswas significantly decreased 45 min after CNO injection (n=8; ⁺p<0.0001,one-way ANOVA). In sham animals, CNO injection did not significantlychange MAP (n=7; one-way ANOVA). FIG. 10B shows changes in the heartrate (HR) in response to CNO injection. 45 min after CNO injection, HRin DREADDs-expressing was significantly decreased (n=8; ⁺p<0.0001,one-way ANOVA), while CNO injection did not significantly change HR insham animals (n=7; one-way ANOVA). The values for both MAP (FIG. 10A)and HR (FIG. 10B) represent the averages of each recorded 20 min priorto CNO injection and 45 min after CNO injection on control days.

FIG. 11 shows that the activation of oxytocin neurons blunts theincrease in blood pressure that occurs with hypoxia/hypercapnia.

FIG. 12 shows changes in MAP from control days to day 21 of CIH/Hexposure. MAP in sham animals significantly increased from day 9 to day21 compared to control day and reached hypertensive levels by day 12(n=7; ⁺p<0.0001, one-way ANOVA). MAP did not significantly increase inDREADDs-expressing OXT neuron activated animals over the 21 days ofCIH/H exposure compared to control days (n=8; one-way ANOVA). MAPsignificantly increased in sham animals compared to DREADDs-expressinganimals from day 12 to day 21 (*p<0.0001, two-way ANOVA with repeatedmeasures).

FIG. 13 shows that the nasal administration of oxytocin to OSA patientsreduces the duration of apnea experienced by the patients.

FIG. 14 shows adverse oxygen desaturations, in percent oxygen, thatoccur with and without the administration of oxytocin to OSA patients.

FIG. 15 shows that the nasal administration of oxytocin to OSA patientsreduces the number of arousals per hour experienced by the patients.

FIG. 16 shows the nasal administration of oxytocin to OSA patientsimproves their sleep quality or sleep satisfaction.

DETAILED DESCRIPTION

Sleep apnea is a common disorder in which an individual have one or morepauses in breathing or shallow breaths while sleeping. The most commontype of sleep apnea is obstructive sleep apnea (OSA). In this condition,the upper airway repetitively collapses or becomes blocked during sleep.This causes shallow breathing or breathing pauses. Breathing pauses orthe duration of apnea can last from a few seconds to minutes. They mayoccur 30 times or more an hour. Typically, normal breathing then startsagain, sometimes with a loud snort or choking sound.

Breathing pauses in individuals with OSA lead to repetitive intermittentperiods of hypoxia/hypercapnia (H/H) during sleep that are accompaniedby arterial oxygen desaturations and increases in arterial carbondioxide levels. OSA is an independent risk factor for the development ofhypertension, coronary artery disease, sudden cardiac death andarrhythmias (Sanchez-de-la-Torre et al. 2013). Patients suffering fromOSA have increases in blood pressure, lower heart rate variability, andreduced baroreflex sensitivity (Carlson et al. 1996; Trimer et al. 2013;Konecny et al. 2014), with chronic impairment in cardiac autonomicfunction i.e., sympathetic hyperactivity and diminished parasympatheticactivity (Trimer et al. 2013).

Continuous positive airway pressure (CPAP) therapy is the most commontreatment for OSA, however this treatment is only modestly effective(Bazzano et al., 2007), not well tolerated by many patients, and its useis often discontinued. Thus, other avenues of treatment are crucial tomitigate the adverse cardiovascular consequences of OSA.

Animal models of OSA based on exposure to chronic intermittent hypoxia(CIH) or hypoxia/hypercapnia (CIH/H) closely mimic OSA in humans(Fletcher et al. 1992; Campen et al. 2005; Kline et al. 2007). While itis known CIH decreases the baroreflex control of heart rate anddiminishes parasympathetic activity to the heart, the exact mechanism ofhow CIH impairs the function of cardiac vagal neurons (CVNs) is notknown. The present disclosure elucidates the mechanism of howhypoxia/hypercapnia experienced during OSA leads to cardiac dysfunctionand provides methods for treating OSA and OSA-induced cardiacdysfunction by administering an effective dose of oxytocin. The presentdisclosure is also based, in part, on the surprising discovery thatadministration of oxytocin to OSA patients also improves sleep qualityexperienced by these patients.

Methods of Treating/Alleviating Obstructive Sleep Apnea

The present disclosure shows for the first time that administration ofoxytocin to OSA patients reduces the duration of apnea experienced bythe OSA patients. Accordingly, the present disclosure provides methodsfor treating OSA in a patient comprising administering to the patient aneffective dose of oxytocin.

In one embodiment, oxytocin is administered intranasally; however, otherroutes of administration such as intravenous, intramuscular,subcutaneous, oral, etc. may also be used. In one embodiment, theeffective dose of oxytocin is about 40 International Units (IU) per day.In one aspect, the effective dose of oxytocin is administered closer tothe sleeping time of the patient, for instance, about 10 minutes, 20minutes, 30 minutes, 40 minutes, 50 minutes, or about 60 minutes priorto the patient going to sleep. In another aspect, oxytocin isadministered within an hour of the patient falling asleep.

In some embodiments, the OSA patient treated with oxytocin may also bereceiving continuous positive airway pressure (CPAP) therapy. CPAPtherapy includes wearing an inhalation system that comprises a nasalmask/piece connected via hose to a small machine that supplies airpressure to keep the airways open and prevent airway occlusion.

In certain embodiments, the effective dose of oxytocin is administeredto the OSA patient receiving CPAP therapy via the CPAP inhalationsystem. For instance, in one embodiment, the disclosure provides methodsfor treating OSA in a patient receiving CPAP therapy, comprisingadministering to the patient at least 40 IU of oxytocin intranasally viathe CPAP inhalation system within an hour of the patient falling asleep.In another embodiment, the disclosure provides methods for treating OSAin a patient receiving CPAP therapy, comprising administering to thepatient an effective dose of oxytocin intranasally via the CPAPinhalation system over the duration of the sleep. For example, in oneembodiment, the effective dose of oxytocin is 40 IU and it isadministered via the CPAP inhalation system at the rate of about 5IU/hour over the sleep period of 8 hours. In another embodiment, theeffective dose of oxytocin is 40 IU and it is administered via the CPAPinhalation system at the rate of about 5.7 IU/hour over the sleep periodof 7 hours. In yet another embodiment, the effective dose of oxytocin is40 IU and it is administered via the CPAP inhalation system at the rateof about 6.6 IU/hour over the sleep period of 6 hours. In yet anotherembodiment, the effective dose of oxytocin is 40 IU and it isadministered via the CPAP inhalation system at the rate of about 8IU/hour over the sleep period of 5 hours.

In OSA patients, the duration of apnea can vary and may last from a fewseconds to minutes. In one embodiment, administration of oxytocin to OSApatients reduces the duration of apnea by at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to the durationprior to the oxytocin treatment.

In one embodiment, administration of oxytocin to the OSA patient reducesor prevents the risk of developing cardiorespiratory diseases in thepatient. The cardiorespiratory diseases that may be prevented oralleviated by administration of oxytocin include, but are not limited tohypertension, cardiac arrhythmias, myocardial ischemia, sudden cardiacdeath and stroke. The present disclosure also shows for the first timethat administration of oxytocin to OSA patients improves sleepsatisfaction and sleep quality in these patients.

CPAP is the most common treatment for OSA; however, many patients do notcomply with CPAP therapy due to problems such as discomfort associatedwith wearing the nasal mask, difficulty tolerating forced air, dry mouthor nose, etc. In one embodiment, administration of oxytocin inaccordance with the present disclosure increases patient compliance withCPAP treatment as oxytocin reduces the duration of sleep apnea andimproves sleep quality.

In certain embodiments, the present disclosure provides methods fortreating OSA patients that are dissatisfied with their current OSAtreatment comprising administering to the patients an effective dose ofoxytocin. Administration of oxytocin in accordance with the presentdisclosure would increase patient compliance with their current OSAtreatment. In one embodiment, patients dissatisfied with their currentOSA treatment include patients receiving CPAP therapy.

Oxytocin

Oxytocin is a nine amino acid cyclic peptide hormone with two cysteineresidues that form a disulfide bridge between positions 1 and 6.Oxytocin is released from the posterior lobe of the pituitary gland andstimulates the contraction of smooth muscle of the uterus during laborand facilitates release of milk from the breast during nursing. Studieshave shown that oxytocin, likely released from a different population ofPVN neurons, may exert a wide spectrum of other biological effectsincluding control of memory and learning processes, and various types ofmaternal and sexual behavior. In addition, oxytocin may participate inthe control of cardiovascular functions, thermoregulation and fluidbalance. Oxytocin is approved by the Food and Drug Administration forintravenous use to induce labor in pregnant women as well as for thetreatment of postpartum hemorrhage. At this time, there are no clinicalor regulatory guidelines for the use of oxytocin in the treatment ofsleep apnea.

The oxytocin peptide for use in the methods described herein can benatural or synthetic, therapeutically or prophylactically active,peptide fragments, peptide analogues, and chemically modifiedderivatives or salts of active peptides. There are processes describedfor the production of oxytocin, see for example U.S. Pat. Nos. 2,938,891and 3,076,797; in addition, oxytocin is commercially available. Avariety of peptide analogues and derivatives are available and otherscan be contemplated for use within the present disclosure and can beproduced and tested for biological activity according to known methods.Oxytocin analogues may be included, but are not limited to,4-threonine-1-hydroxy-deaminooxytocin, 4-serine, 8-isoleucine-oxytocin,9-deamidooxytocin, 7-D-proline-oxytocin and its deamino analog,(2,4-diisoleucine)-oxytocin, deamino oxytocin analog,1-deamino-1-monocarba-E12-Tyr(OMe)]-OT(dCOMOT), carbetocin, 4-threonine,7-glycine-oxytocin (TG-OT), oxypressin, deamino-6-carba-oxytoxin (dC60),L-371,257 and the related series of compounds containing anortho-trigluoro-ethoxyphenylacetyl core such as L-374,943. Oxytocinpeptides for use within the present disclosure can be peptides that areobtainable by partial substitution, addition, or deletion of amino acidswithin a naturally occurring or native peptide sequence. Peptides can bechemically modified, for example, by amidation of the carboxyl terminus(—NH₂), the use of D amino acids in the peptide, incorporation of smallnon-peptidyl moieties, as well as the modification of the amino acidsthemselves (e.g. alkylation or esterification of side chain R-groups).Such analogues, derivatives and fragments should substantially retainthe desired biological activity of the native oxytocin peptide.

Routes and Ranges of Administration of Oxytocin

The route of administration of oxytocin will depend upon the age, weightand/or the physical condition of the patient and timing ofadministration. In various embodiments, oxytocin can be administered toa patient nasally, orally, intravenously, intradermally, transdermally,subcutaneously, intramuscularly, topically, intrathecally andintracerebroventricularly.

In one embodiment, oxytocin is administered nasally or intranasally.Intranasal delivery has a number of advantageous features includingcomparatively high bioavailability, rapid kinetics of absorption andavoidance of a first-pass effect in the liver. In regard to patientcompliance and ease of use, intranasal administration provides a simple,rapid and non-invasive mode of application. Oxytocin or a pharmaceuticalcomposition comprising oxytocin can be administered to the nasal cavityas a powder, a granule, a solution, a cream, a spray, a gel, a film, anointment, an infusion, a drop or a sustained-release composition. In oneembodiment, oxytocin or a pharmaceutical composition comprising oxytocincan be administered intranasally using the CPAP inhalation system. Inthese embodiments, oxytocin can be vaporized or aerosolized and providedvia the CPAP inhalation system continuously or at regular intervals.

A therapeutically effective dose of oxytocin will depend upon the age,weight and/or the physical condition of the patient and route ofadministration. In some embodiments, the effective dose of oxytocin mayrange from about 10-60 IU, 20-50 IU, or 25-45 IU per day. In someembodiments, the effective dose of oxytocin is about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, or 60 IU per day. In one embodiment, the effectivedose of oxytocin is at least 40 IU per day. In another embodiment, theeffective dose of oxytocin is at least 40 IU every other day. In yetanother embodiment, the effective dose of oxytocin is about 25 or 30 IUper day. In yet another embodiment, the effective dose of oxytocin is atleast 25 or 30 IU every other day. In some embodiments, the effectivedose of oxytocin is administered weekly. In some embodiments, thetherapeutically effective dose of oxytocin is not 10 units administeredat the time of sleep or over a 7 hour period. In some embodiments, thetherapeutically effective dose of oxytoxin is not administeredintramuscularly or intravenously.

The effective dose of oxytocin can be administered in a single dose orin multiple doses, for example, dosages can be administered two, three,four, five, six, seven, eight, nine or ten times daily. In oneembodiment, the effective dose of oxytocin is 40 IU and is administeredas a one-time nasal spray shortly before sleeping or within an hour ofthe patient falling asleep.

Preferably, oxytocin is administered closer to the sleeping time or overthe duration of sleep. For instance, in one embodiment, the effectivedose of oxytocin can be administered about 10, 20, 30, 40, 50 minutes orabout an hour prior to sleeping. In another embodiment, the effectivedose of oxytocin can be administered within about 10, 20, 30, 40, 50minutes or about an hour of the patient falling asleep. In someembodiments, the effective dose of oxytocin is administered as a singledose via the CPAP inhalation system within an hour of the patientfalling asleep. In some other embodiments, the effective dose ofoxytocin is administered via the CPAP inhalation system over theduration of sleep either continuously or at regular intervals.

Treatment of OSA Induced Cardiorespiratory Diseases

OSA represents a major, yet poorly understood cardiovascular risk factorin ˜24% of males and 9% of females within the US population. Severe OSAincreases cardiovascular mortality 4 fold, and even when corrected forother risk factors increases cardiovascular mortality 3 fold. OSA canplay a role in both the initiation and progression of severalcardiovascular/cardiorespiratoty diseases including sudden death,hypertension, arrhythmias, myocardial ischemia and stroke.

Prior studies by the inventors have shown that activation ofoxytocin-positive PVN neurons decreases resting heart rate (HR) andblood pressure (BP). However, the activation of oxytocin-positive PVNneurons does not necessarily indicate that oxytocin is the mediator ofthe observed decrease in HR and BP. These oxytocin neurons secrete manychemicals at their synapse, including fast neurotransmitters. Prior workhas shown these oxytocin neurons release the fast neurotransmitterglutamate, and activation of postsynaptic NMDA and AMPA glutamatereceptors are primarily responsible for the excitation of cardiac vagalneurons (Pinol et al., 2014). While anatomical work has shown thepresence of peptides, including oxytocin, in the projections from thePVN to parasympathetic nuclei, prior work has not demonstrated therelease of peptides either in replacement of, or in addition to, fastneurotransmitters (such as glutamate), and electrophysiological studiesto date have only demonstrated release of glutamate and activation offast ligand gated receptors in these pathways (Pinol et al., 2014). Thepresent disclosure shows for the first time that activation ofoxytocin-positive PVN neurons evokes endogenous release of oxytocin thatis diminished with CIH/H, and that selective activation ofoxytocin-secreting PVN neurons during CIH/H exposure both restoresoxytocin release and prevents the CIH/H-elicited elevations in BP tohypertensive levels that occurs in untreated animals. That is, thepresent disclosure shows for the first time that there is release ofoxytocin from oxytocin-secreting PVN neurons, and that this release ofoxytocin is cardio-protective. The present disclosure also shows thatadministration of oxytocin in OSA patients decreases the duration ofapnea and the arterial oxygen desaturation that occurs duringhypoxia/hypercapnia episodes in OSA patients further confirming thecardio-protective role of oxytocin in OSA patients.

In one embodiment, the present disclosure provides a method for treatinga patient suffering from OSA induced cardiorespiratory disease,comprising administering to the patient an effective dose of oxytocin.In one embodiment, the effective dose of oxytocin is 40 IU and isadministered intranasally within an hour of the patient falling asleep.OSA induced cardiorespiratory diseases that may be treated in accordancewith the present disclosure include, but are not limited to,hypertension, cardiac arrhythmias, myocardial ischemia, sudden cardiacdeath and stroke. The aforementioned method of treating a patientpopulation for cardiorespiratory disease via oxytocin treatment is verysurprising given the fact that previous electrophysiological studieshave only demonstrated release of glutamate and activation of fastligand gated receptors in the PVN to parasympathetic nuclei pathways.See, supra, FIG. 1 and Pinol et al., 2014. The inventors have thereforediscovered a unique method of treating a heretofore unidentified patientpopulation.

In one aspect, the present disclosure provides a method for treating apatient suffering from OSA induced hypertension, comprisingadministering to the patient an effective dose of oxytocin. In oneembodiment, the effective dose of oxytocin is at least 40 IU and isadministered intranasally within an hour of the patient falling asleep.

In another aspect, the present disclosure provides a method for treatinga patient suffering from OSA and/or OSA induced hypertension andcompromised sleep quality, comprising activating oxytocin-secreting PVNneurons in the patient.

In some embodiments, the patient being treated with oxytocin for OSAinduced cardiorespiratory diseases may also be receiving CPAP therapy.

In one embodiment, administration of oxytocin reduces the hypertensionexperienced by the patient. In another embodiment, administration ofoxytocin decreases the heart rate and/or blood pressure of the OSApatient compared to the heart rate and/or blood pressure prior to thetreatment with oxytocin. In some embodiments, administration of oxytocindecreases the heart rate and/or blood pressure of the OSA patient by atleast 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to theheart rate and/or blood pressure prior to the treatment with oxytocin.

In one embodiment, administration of oxytocin prevents the heart rateand/or blood pressure of the OSA patient from increasing, i.e. oxytocinmaintains the heart rate and/or blood pressure to the levels normallyfound in the patient.

In certain embodiments, administration of oxytocin to a patientsuffering from OSA induced cardiorespiratory disease reduces the oxygendesaturation experienced by the patient during apnea episodes. The term“oxygen desaturation” as used herein refers to a decrease in bloodoxygen levels from a normal value of ˜95 percent. In one embodiment,administration of oxytocin reduces the oxygen desaturation experiencedby the patient by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to the oxygen desaturationprior to the treatment with oxytocin.

Methods of Improving Sleep Quality in OSA Patients

Previous studies have shown that different routes of administration ofoxytocin, for example, externally icy administered oxytocin representeda stressful event and induced arousal and did not promote sleep. Forexample, Lancel et al. (Regulatory Peptides, 2003, 114: 145-152) showedin rats under basal, stress-free conditions, endogenous oxytocinpromotes sleep whereas acute icy infusion of oxytocin delayed sleeponset latency, which resulted in a transient reduction of non-REMS andREMS, and augmented high-frequency activity in the electroencephalogram(EEG) within non-REMS. Lancel et al. concluded that external icyadministration of oxytocin reflected a condition of stress and wasaccompanied by behavioral arousal and increase vigilance.

Sleep apnea is a stressful event as it represents a strong adversechallenge to the cardiorespiratory system that impedes the breathingprocess necessary to supply oxygen to the rest of the body. The presentdisclosure, however, unexpectedly found that, in contrast to previousstudies, external administration of oxytocin to OSA patients improvedsleep quality or sleep satisfaction in these patients. Accordingly, inone embodiment, the present disclosure provides methods for improvingsleep quality in a patient suffering from OSA, comprising administeringto the patient an effective dose of oxytocin. In one embodiment, theeffective dose of oxytocin is at least 40 IU and is administeredintranasally within an hour of the patient falling asleep. Theaforementioned method of improving sleep quality is counterintuitive inview of references such as the above cited Lancel, et al. and representsa significant advancement in sleep medicine.

In one embodiment, administration of oxytocin to OSA patients decreasesthe number of arousals per hour experienced by the patient leading tobetter sleep. “Arousals” are defined as “abrupt changes in EEGfrequency, which last for ≥3 seconds, and are preceded by at least 10seconds of EEG sleep.” Frequency of arousals is denoted by “arousalindex” (arousals/hour) and correlates positively with feelings ofnon-refreshing sleep, i.e. higher the arousal index, more likely thepatient will complain of non-refreshing sleep. In some embodiments,administration of oxytocin to OSA patients decreases the number ofarousals per hour experienced by the patient by at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, or 50% compared to the number of arousals perhour prior to the treatment with oxytocin.

In some embodiments, the quality of sleep or sleep satisfaction in OSApatients is assessed by asking the patients to rank their responses onthe scale of 1-5 to a set of empirical factors. For instance, in oneembodiment, a set of empirical factors includes the following parametersor questions:

-   -   I feel more refreshed than usual this morning    -   My quality of sleep last night was better than usual    -   I slept deeper than usual last night    -   I woke up fewer times than usual last night    -   I slept longer than usual last night    -   I feel better overall than usual this morning        The patients are asked to rank their response to the above        empirical factors on the scale of 1-5 as follows:    -   1—Strongly disagree    -   2—Slightly disagree    -   3—Neither agree nor disagree    -   4—Slightly agree    -   5—Strongly agree        Based on their responses, a sleep score for each patient is        calculated. In one embodiment, administration of oxytocin to OSA        patients leads to improvement in empirical factors or sleep        score indicative of sleep quality in the OSA patient.

In one embodiment, administration of oxytocin to OSA patients decreasesthe duration of apnea experienced by the patients leading to bettersleep. In one embodiment, administration of oxytocin to OSA patientsreduces the duration of apnea by at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to the duration prior tothe oxytocin treatment.

Pharmaceutical Compositions

While it is possible to administer oxytocin alone, there may besituations wherein it is advantageous to present it as part of apharmaceutical composition. Thus, in some aspects of the presentinvention, oxytocin is administered as a pharmaceutical composition. Thepharmaceutical composition can comprise oxytocin at a therapeuticallyeffective dose together with one or more pharmaceutically acceptablecarriers and optionally other ingredients. A suitable carrier is onewhich does not cause an intolerable side effect, but which allowsoxytocin to retain its pharmacological activity in the body. A carriermay also reduce any undesirable side effects of oxytocin. A suitablecarrier should be stable, i.e., incapable of reacting with otheringredients in the formulation. A suitable carrier should have minimalodor or fragrance or a positive (pleasant) odor. A suitable carriershould not irritate the mucosa, epithelium, underlying nerves or providea health risk. It may be an accepted transcutaneous or percutaneouscarrier or vehicle, because any carrier that can effectively penetratethe stratum corneum of the skin should be highly efficacious in not onlypenetrating mucosa, but also allowing rapid absorption of substancesinto the submucosal tissues, nerve sheaths and nerves.

Suitable nontoxic pharmaceutically acceptable carriers will be apparentto those skilled in the art of pharmaceutical formulations. Also seeRemington: The Science and Practice of Pharmacy, 20th Edition,Lippincott, Williams & Wilkins (2000). Typical pharmaceuticallyacceptable carriers include, but are not limited to, mannitol, urea,dextrans, lactose, potato and maize starches, magnesium stearate, talc,vegetable oils, polyalkylene glycols, ethyl cellulose,poly(vinylpyrrolidone), calcium carbonate, chitosan, ethyl oleate,isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin,potassium carbonate, silicic acid, and other conventionally employedacceptable carriers. Other carriers include, but are not limited to,phosphatidylcholine, phosphatidylserine, and sphingomyelins.

The choice of a suitable carrier will depend on the exact nature of theparticular formulation desired, e.g., whether the drug is to beformulated into a liquid solution (e.g., for use as drops, for use in aninjection, as a spray or impregnated in a nasal tampon, or otheragent-impregnated solid), a suspension, a ointment, a film or a gel. Ifdesired, sustained-release compositions, e.g. sustained-release gels,films, transdermal patchs, etc. can be readily prepared. The particularformulation will also depend on the route of administration. In oneembodiment, a composition comprising oxytocin can be administered to thenasal cavity as a powder, a granule, a solution, a cream, a spray, agel, a film, an ointment, an infusion, a drop or a sustained-releasecomposition.

To enhance delivery into or across the nasal mucosal surface and/orabsorption of a pharmaceutical composition comprising oxytocin, anabsorption-enhancing agent can be included in the formulation. Theseenhancing agents may enhance the release or solubility (e.g., from aformulation delivery vehicle), diffusion rate, penetration capacity andtiming, uptake, residence time, stability, effective half-life, peak orsustained concentration levels, clearance and other desired mucosaldelivery characteristics (e.g., as measured at the site of delivery) ofthe composition. Absorption enhancing compounds may include, but are notlimited to, surfactants, bile salts, dihydrofusidates, bioadhesiveagents, phospholipid additives, mixed micelles, liposomes, or carriers,alcohols, enamines, cationic polymers, NO donor compounds, long-chainamphipathic molecules, small hydrophobic penetration enhancers; sodiumor a salicylic acid derivatives, glycerol esters of acetoacetic acid,cyclodextrin or beta-cyclodextrin derivatives, medium-chain fatty acids,chelating agents, amino acids or salts thereof, N-acetylamino acids orsalts thereof, mucolytic agents, enzymes specifically targeted to aselected membrane component, inhibitors of fatty acid synthesis andinhibitors of cholesterol synthesis.

This disclosure is further illustrated by the following additionalexamples that should not be construed as limiting. Those of skill in theart should, in light of the present disclosure, appreciate that manychanges can be made to the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the disclosure.

EXAMPLES Example 1

Labeling of CVNs: To obtain electrophysiological recordings from CVNs,neonatal Sprague-Dawley rats (postnatal days 2-5, Hilltop Laboratoryanimals Inc, Scottdale, Pa., USA) were anesthetized using hypothermia bycooling to approximately 4° C. A right thoracotomy was performed andretrograde tracer X-Rhoda-mine-5-(and-6)-isothiocyanate (Invitrogen,USA) was then injected into the fat pads at the base of the heart toretrogradely label CVNs (Mendelowitz & Kunze 1991). The animals werethen allowed to recover until they were 3-4 weeks old.

Telemetry Implantation: To record blood pressure and heart rate, maleSprague-Dawley rats, 3-4 weeks of age, were anesthetized usingisoflurane (2-4%) and a HD-X11 pressure transmitter was implanted (DataSciences International, St Paul, Minn., USA) with its cathether insertedinto the abdominal aorta to record pressure and EKG leads were attachedsubcutaneously to obtain EKG recordings and heart rate. All rats withtelemetry devices were allowed 7-14 days to recover from transmitterimplantation surgery before any measurements were recorded. Bloodpressure and heart rate were recorded via radio-frequency signalsobtained through the Ponemah data acquisition system (Data SciencesInternational). Baseline recordings of blood pressure and heart ratewere obtained for 3 days prior to CIH/H exposure. Prior to, and during,the 28 day CIH/H exposure period daily baseline recordings of bloodpressure and heart rate were recorded.

Air or CIH/H exposure: Animals were exposed to repetitive cycles of 3minutes of mild H/H (6% O₂+5% CO₂+89% N₂) followed by 3 minutes ofnormoxia (21% O₂+79% N₂), repeated for 10 times per hour, 8 hr/day, for3 or 4 weeks. The animals were exposed to CIH/H for 8 hours during lightphase and to normal air during the remaining 16 hr. Unexposed animalsthat were exposed to normal air (21% O₂+79% N₂), were placed adjacent tothe chambers during the exposure period to undergo similar handling,general lab conditions, and background noise as the CIH/H group.

In vitro brainstem slice preparation: The methodology described by Yeand colleagues (Ye et al. 2006) was used to obtain viable brainstemslices from mature animals. According to this method, glycerol baseartificial cerebrospinal fluid (aCSF) was used for cardiac perfusion andbrainstem slicing. Glycerol-based aCSF contained (in mM): 252 glycerol,1.6 KCl, 1.2 NaH₂PO₄, 1.2 MgCl, 2.4 CaCl₂, 26 NaHCO₃, and 11 glucose.Immediately following air or CIH/H exposure for 4 weeks, rats wereanaesthetized using isoflurane and placed on ice. Glycerol aCSF (4° C.,pH: 7.4, bubbled with 95% O₂−5% CO₂) was perfused transcardially at aspeed of ˜10 ml/min after which the brain was quickly removed, glued onto a stage using 2% low melt agarose and placed in a vibrotomecontaining glycerol aCSF. Brainstem slices (330 μm thickness) containingeither DMNX or NA or brainstem slices containing dorsal motor nucleus(DMV) and channelrhodopsin (ChR2)-containing PVN fibers were obtainedand briefly placed in a solution with following composition (in mM): 110N-methyl-d-glucamine (NMDG), 2.5 KCl, 1.2 NaH₂PO₄, 25 NaHCO₃, 25glucose, 110 HCl, 0.5 CaCl₂, and 10 MgSO₄ equilibrated with 95% O₂ and5% CO₂ (pH 7.4) at 34° C. for 1 5 min. NMDG based aCSF was used to helpslices recover and to maintain viable brainstem slices forelectrophysiological recordings (Zhao et al. 2011). The slices were thenmounted in a recording chamber constantly perfused with a normal aCSFwith following composition (in mM): 125 NaCl, 3 KCl, 2 CaCl₂, 26 NaHCO₃,5 glucose and 5 HEPES; oxygenated with 95% O₂−5% CO₂ (pH-7.4) andallowed to recover for at least 30 minutes before an experiment wasperformed.

Electrophysiological recordings: CVNs in NA and DMNX were identified bythe presence of fluorescent tracer rhodamine and imaged usingdifferential interference contrast optics and infrared illumination.Whole cell voltage clamp recordings from CVNs were done using Axopatch200B and pClamp 8 software (Axon Instruments, Union city, USA), at aholding voltage of −80 mV at room temperature. The patch pipettes (2.5-5MΩ) were filled with a solution consisting (in mM) of KCl (150), MgCl₂(4), EGTA (10), Na-ATP (2) and HEPES (10) or K-gluconic acid (150),HEPES (10), EGTA (10), MgCl₂ (1) and CaCl₂ (1) at a pH of 7.3 forrecording inhibitory or excitatory events respectively. For PVN studies,forebrain slices were used for electrophysiology recordings.

Drugs were focally applied to CVNs using a pneumatic picopump pressuredelivery system. GABAergic inhibitory post synaptic currents (IPSCs)were isolated by focal application of solution containing strychnine (1μM, glycine receptor antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX, 50 μM, non-NMDA receptor antagonist) andD-2-amino-5-phosphonovalerate (AP5, 50 μM, NMDA receptor antagonist),with the puffer pipette positioned near the patched neuron. GlycinergicIPSCs were isolated by including gabazine (25 μM, GABA-A receptorantagonist), CNQX, and AP5 in the puffer pipette. The puffer pipette wasfilled with gabazine and strychnine to isolate glutamatergic excitatorypostsynaptic currents (EPSCs).

Acute H/H: The respective EPSCs or IPSCs were recorded in controlconditions for 5 min in the presence of aCSF equilibrated with 95% O₂₊₅%CO₂. Brainstem slices containing CVNs were exposed to H/H by superfusingthe aCSF equilibrated with 85% N₂+6% O₂+9% CO₂ for 10 min. Gabazine,strychnine, or CNQX, and AP5 were applied at the end of each experimentto confirm the targeted isolation of GABAergic, glycinergic, orglutamatergic activity, respectively. Each slice was exposed to hypoxiaonly once limiting the experiments to only one CVN per slice of tissue.Gabazine, strychnine, CNQX, and AP5 were obtained from Sigma Aldrich(St. Louis, Mo., USA).

Viral vectors, plasmids, and promoter constructs: Lentiviral plasmidspLenti-Syn-hChR2(H134R)-EYFP-WPRE, packaging plasmid pCMV-ΔR8.74 andenvelope plasmid pMD2.G were all kindly donated by K. Deisseroth(Stanford University, Stanford, Calif., USA). ThepLenti-Syn-hChR2(H134R)-EYFP-WPRE viral vector was produced according tocustomary protocols as described before (Wsol et al., 2009).

A rat minimal oxytocin (OXT) promoter element from −530 bp to +33relative to the origin of transcription of the OXT gene (UCSC genomebrowser on rat November 2004 assembly; chr3:118,193,690 to 118,194,252)was synthesized de novo and flanked by multiple cloning sites(Genscript, Piscataway, N.J.) (McCann et al., 2002; Petersson et al.,1996). The rAAV1-OXT-Cre was produced using the OXT promoter fragment inthe following way (MIT, Viral Gene Transfer Core): pFB-AAV-OXT promoterCre was created by cloning the OXT promoter into V032 by excising theOXT promoter/pUC57 with XbaI (5′) and AgeI (3′) and cloning it into V032cut with SpeI (5′) and AgeI (3′). Then Cre was added by cutting Cre outof pBS185 with XhoI (5′) and MluI-blunt (3′) and moving it intopFB-AAV-OXT cut with XhoI (5′) and Asp718-blunt (3′). To achieve robustand highly selective expression of designer receptors exclusivelyactivated by designer drugs (DREADDs) in PVN OXT neurons, the reporterviral vector AAV2-hSyn-DIO-hM₃D(G_(q))-mCherry (UNC, Gene TherapyCenter, Vector Core Services) was co-injected with AAV-OXT-Cre.Expression of these Cre-dependent vectors will only be initiated inneurons selectively expressing Cre as they contain silencingdouble-floxed inverse open reading frames (Sawchenko and Swanson, 1982).OXT receptors, as well as the red fluorescent calcium indicator, wereexpressed in Chinese hamster ovary (CHO) cells as previously described(Gamer and Buchel, 2012).

Stereotactic injections: Stereotactic injections were performed aspreviously described (Bradley and Floras, 2009; Parish and Somers, 2004;Pinol et al. 2014). pLenti-Syn-hChR2(H134R)-EYFP-WPRE (90-100 nl) wasinjected for experiments involving activation of channelrhodopsin(ChR2)-expressing PVN fibers, while 20-30 nl of viral vectorsrAAV1-OXT-Cre and AAV2-hSyn-DIO-hM₃D(G_(q))-mCherry at a 1:2 ratio wasinjected for PVN OXT neuron activation.

Calcium imaging in OXT receptor-expressing CHO cells: Visualization ofCHO cells expressing OXT receptors and the calcium indicator, as well asChR2-containing PVN fibers, were performed on a confocal microscopesystem as previously described (Gamer and Buchel, 2012). To examineactivation of OXT receptors upon optogenetic activation ofChR2-expressing PVN fibers in the DMV, CHO cells were pipetted onto thedorsal motor nucleus (DMV) of brain stem slices from animals previouslyinjected with pLenti-Syn-hChR2(H134R)-EYFP-WPRE into the PVN.OXT-sensitive CHO cells within the boundaries of the DMV and in closeapposition to ChR2-containing PVN fibers (7.5±0.5 μm) were used forexperiments.

Daily activation of DREADDs: 1-2 weeks after telemetry implantation,DREADDs receptors on PVN OXT neurons were activated daily byintraperitoneal (IP) injection of clozapine-n-oxide (CNO, 1.0 mg/kg). Toinvestigate the effects of CNO on resting BP and HR in bothDREADDs-expressing and sham animals, 3 days prior to CIH/H exposure(control days) baseline BP and HR values were recorded for a 20 minperiod before CNO injection. Animals from each group then received an IPinjection of CNO, BP and HR were recorded for 1 hr, and the animals thenunderwent exposure to air for 8 hr to achieve acclimation to thechambers. During the 21 days of CIH/H exposure, baseline recordings ofBP and HR were obtained before CNO injection, recorded for 1 hr post CNOinjection, and for the entire duration of CIH/H exposure.

Data Analysis for studies directed to the effects of CIH/H on CVNs:Synaptosoft software (version 6.0.3; Synaptosoft, Decatur, Ga.) was usedto analyze the synaptic events recorded from CVNs. Threshold value wasset to the root mean square of noise levels multiplied by 5. Thefrequency and amplitudes of synaptic currents were grouped in 10 secbins and averaged for 2 min at the end of control and H/H. The data werepresented as mean±SEM. To examine the chronic changes in blood pressureand heart rate over the 28 day CIH/H exposure, daily values recordedbefore each CIH/H exposure were statistically analyzed by One-wayrepeated-measures analysis of variance (One-way ANOVA) followed byBonferroni's multiple comparison test. Students unpaired t-test was usedto compare statistical significance between unexposed and CIH/H exposedgroups. For acute H/H evoked blood pressure and heart rate responsesduring CIH/H exposure and in-vitro experiments utilizing differentconditions in the same CVN, Student's paired t-test was used to test thesignificance using Graphpad Prism 5 software (La Jolla, Calif., USA).Data with p<0.05 was considered significant; in the figures, * denotesp<0.05, ** denotes p<0.01, *** denotes p<0.001.

Data Analysis for studies directed to the effects of CIH/H on PVNs:Calcium responses in CHO cells were grouped into 0.5 sec bins for atotal of 10 bins with the 3^(rd) bin (time 0) representing the time ofChR2-expressing PVN fiber activation. Results are presented as percentcontrol and statistically compared with data from the same experimentusing a one-way ANOVA with repeated-measures followed by Bonferroni'smultiple comparison test. For those experiments comparing CHO cellresponses in unexposed brainstem tissue to CIH/H exposure, a one-wayANOVA was used. Data with p<0.05 was considered significant.

Changes in the action potential firing frequency were determined usingthe MiniAnalysis version 6.0.3 software (Synaptosoft, Decatur, Ga.) andgrouped into 5 min bins for a total of 12 bins with the 2^(nd) binrepresenting the start of CNO application. Results are presented asmeans±SE and statistically compared with control data from the sameexperiment using a one-way ANOVA with repeated-measures followed byBonferroni's multiple comparison test for all experiments. Data withp<0.05 were considered significant.

To examine the effects of CNO on and the chronic changes to meanarterial blood pressure (MAP) over the 21 days of CIH/H exposure, valueswere recorded before and after CNO injection on control days prior toCIH exposures, and days 1, 3, 6, 9, 12, 15, 18, and 21 of CIH/H. Changeswithin the DREADDs-expressing animals and the sham animals werestatistically analyzed by one-way ANOVA with repeated-measures followedby Bonferroni's multiple comparison test. To examine the statisticalchanges in MAP between the two groups of animals, a two-way ANOVA withrepeated-measures followed by Bonferroni's multiple comparison test wasused. Data with p<0.05 were considered significant.

Software used for all statistical analysis of the data included GraphpadPrism 4.01 (Graphpad Software, San Diego, Calif.), MicroCal Origin 7.0(OriginLabs Corp, Nothhampton, Mass.) and Microsoft Excel (MicrosoftCorp., Redmond, Wash.).

Example 2: Effect of CIH/H on Blood Pressure

Adult rats (4 weeks old) were exposed to CIH/H for 8 hours and to normalair during the remaining 16 hours for 3 weeks as explained above. Bloodpressure and heart rate was examined before and throughout 28 days ofCIH/H exposure. After 4 weeks of CIH/H, systolic and diastolic pressureincreased to hypertensive levels (from a systolic pressure of 105±4.0mmHg at the onset of CIH/H to 144±3.0 mmHg after 28 days of CIH/H, n=6;p<0.05; One way ANOVA, and diastolic pressure increased from 77±1.0 mmHgto 110±5.0 mmHg after 28 days of CIH/H, n=6; p<0.05; One way ANOVA), seeFIG. 2A.

Example 3: Acute H/H Evoked Blood Pressure and Heart Rate Responses atthe Start and End of CIH/H Exposure

At the beginning of the 28 days of CIH/H exposures, during a singleexposure to H/H, heart rate decreased by 25% (438±15 beats/min innormoxia and 325±21 beats/min in acute H/H; n=6; p<0.05; paired t-test),and this decrease in heart rate occurred without significant changes inblood pressure (99±2 mmHg in normoxia and 97±3 mmHg in acute H/H; n=6;p>0.05; paired t-test). However, at the end of 4 weeks of CIH/Hexposure, acute H/H evoked a significant increase in blood pressure(112±7 mmHg in normoxia and 123±5 mmHg in acute H/H; n=6; p<0.01; pairedt-test) while there were no significant changes in heart rate (389±23beats/min in normoxia and 353±27 beats/min in acute H/H; n=6; p>0.05;paired t-test), see FIGS. 2B and 2C.

Example 4: Actions of CIH/H on Inhibitory Neurotransmission to CVNs

GABAergic and glycinergic IPSCs were examined from CVNs both in the NAand DMNX of the brainstem from unexposed and CIH/H animals. In unexposedanimals, the frequency of both GABAergic (7.9±1.2 Hz, n=48 in NA and3.5±0.3 Hz, n=20 in DMNX; p<0.05; Unpaired t-test) and glycinergic(4.4±0.6 Hz, n=29 in NA and 1.8±0.2 Hz, n=27 in DMNX; p<0.001; Unpairedt-test) IPSCs in NA CVNs was greater than that in DMNX CVNs, see FIGS.3C, 3F, 4C, and 4F. In addition, the amplitude of glycinergic IPSCs inCVNs of DMNX was significantly less than that of NA (58.6±9.8 pA, n=29in NA and 23.6±1.5 pA, n=27 in DMNX; p<0.01; Unpaired t-test). Theamplitudes of GABAergic IPSCs in NA and DMNX CVNs were not different(44.0±2.5 pA, n=48 in NA CVNs and 46.6±4.3 pA, n=20 in DMNX CVNs;p>0.05).

CIH/H exaggerated the frequency of GABAergic (but not glycinergic) IPSCsin NA CVNs, whereas glycinergic (but not GABAergic) IPSC frequency wasincreased in DMNX CVNs following CIH/H. The frequency of GABAergic IPSCsrecorded from NA CVNs of CIH/H exposed animals was 49% greater than thatin unexposed animals (7.9±1.2 Hz, n=48 in unexposed and 11.8±1.3 Hz,n=51 in CIH/H exposed; p<0.05; unpaired t-test), FIGS. 3A-3C. In DMNX,no change in GABAergic IPSC frequency to CVNs was observed betweenunexposed and CIH/H exposed animal groups (3.5±0.3 Hz, n=20 in unexposedand 4.5±0.7 Hz, n=25 in CIH/H exposed; p>0.05; unpaired t-test), FIGS.3D-3F. The amplitude of GABAergic IPSCs to CVNs of NA and DMNX inunexposed group was not different from that of CIH/H exposed group.

With respect to glycinergic IPSCs to CVNs, their frequency andamplitudes in CIH/H and unexposed groups were not different in NA CVNs.However, in DMNX CVNs, the frequency of glycinergic IPSCs from CIH/Hgroup was 50% greater compared to unexposed group (1.8±0.2 Hz, n=27 inunexposed and 2.7±0.4 Hz, n=23 in CIH/H exposed; p<0.05; unpairedt-test); see FIGS. 4A-4F.

Example 5: Actions of CIH/H on Excitatory GlutamatergicNeurotransmission to CVNs

The amplitude of EPSCs in NA CVNs was significantly less than theamplitude of EPSCs in DMNX CVNs (18.0±1.8 pA, n=28 in NA and 34.1±1.9pA, n=19 in DMNX; p<0.001; Unpaired t-test). CIH/H significantly reducedthe frequency of glutamatergic EPSCs in CVNs in both NA (4.0±0.4 Hz,n=28 in unexposed and 2.7±0.3 Hz, n=24 in CIH/H exposed; p<0.01;unpaired t-test) and DMNX (4.1±0.3 Hz, n=17 in unexposed and 2.3±0.3 Hz,n=18 in CIH/H exposed; p<0.001; unpaired t-test) compared to unexposedgroup, see FIGS. 5C and 5G-. CIH/H also reduced the amplitude of EPSCsin DMNX, but not NA, CVNs (34.1±3.2 pA, n=17 in unexposed and 25.8±2.5pA, n=18 in CIH/H exposed; p<0.05; unpaired t-test), FIGS. 5D and 5H.

Example 6: Effect of Acute H/H on Inhibitory Neurotransmission to CVNsin Unexposed Animals

GABA: In unexposed animals acute exposure to H/H inhibited the frequencyof GABAergic IPSCs by 40% and 60% in the NA and DMNX CVNs respectively(NA CVNs: 6.3±1.0 Hz in control and 3.7±0.5 Hz in H/H; n=14; p<0.05;paired t-test, DMNX CVNs: 3.4±0.5 Hz in control and 1.3±0.3 Hz in H/H;n=9; p<0.001; paired t-test), see FIGS. 6B and 6E. In addition, H/Hinhibited the amplitude of GABAergic IPSCs in DMNX CVNs (52.5±3.7 pA incontrol and 42.4±3.5 pA in H/H; n=9; p<0.05; paired t-test) but not inCVNs within the NA, see FIGS. 6C and 6F.

Glycine: Acute H/H inhibited the frequency of glycinergic IPSCs in DMNXCVNs by 50% (2.0±0.3 Hz in control and 1.0±0.2 Hz in H/H; n=12; p<0.01;paired t-test). However the frequency and amplitude of glycinergic IPSCsin NA CVNs were unaltered by acute H/H, see FIGS. 7A-7F.

Example 7: Effect of Acute H/H on Inhibitory Neurotransmission to CVNsin CIH/H Exposed Animals

GABA: Similar to the responses in the unexposed group, in animalsexposed to CIH/H acute H/H inhibited the frequency of GABA IPSCs in DMNXCVNs by 60% (4.5±1.6 Hz in control and 1.3±0.3 Hz in H/H; n=11; p<0.05;paired t-test), see FIG. 6K. In contrast, in CIH/H animals the GABAergicresponses to acute H/H on NA CVNs was abolished (7.1±1.2 Hz in controland 7.2±1.5 Hz in H/H; n=13; p>0.05; paired t-test), see FIGS. 6G-6I.

Similar to the responses in the unexposed animal group, in animalsexposed to CIH/H acute H/H reduced the amplitude of GABA IPSCs in DMNXCVNs (42.3±4.5 pA in control and 33.8±3.1 pA in H/H; n=11; p<0.05;paired t-test) but not in NA CVNs, see FIGS. 6I and 6L.

Glycine: Unlike the unexposed animals, in animals exposed to CIH/H acuteH/H significantly increased the frequency of glycinergic IPSCs in NACVNs by 40%, without any significant changes in glycinergic IPSCamplitude (5.5±0.9 Hz in control and 7.8±0.9 Hz in H/H; n=12; p<0.05;paired t-test), see FIGS. 7H and 7I. In animals exposed to CIH/H, acuteH/H inhibited the frequency of glycinergic IPSCs in DMNX CVNs by 25%;see FIG. 7K.

Example 8: Effect of Acute H/H on Glutamatergic Neurotransmission toCVNs of Unexposed and CIH/H Exposed Animals

Acute H/H had no effect on the frequency or amplitude of glutamatergicEPSCs to CVNs in NA and DMNX in both unexposed and CIH/H exposed animals(Data not shown).

Example 9: Selectivity and In Vitro Activation of Excitatory DREADDs inPVN OXT Neurons

Selective excitatory DREADDs expression in PVN OXT neurons was achievedwith injection into the PVN of two viral vectors, one expressing Creunder an OXT promoter (rAAV1-OXT-Cre), and the other a Cre-dependentvector expressing excitatory hM₃D(G_(q)) DREADDs(AAV2-hSyn-DIO-hM₃D(G_(q))-mCherry). Immunohistochemical analysisconfirmed that this viral expression system elicited high (83.1±2.1%)selectivity for DREADDs expression in PVN OXT neurons. The responsesupon activation of DREADDs in PVN OXT neurons was assessed in vitrousing the whole-cell patch clamp method. The action potential firingfrequency of DREADDs-expressing PVN neurons significantly increasedwithin 5 min of CNO application (from 0.19±0.05 Hz to 0.75±0.14 Hz; n=7;*p<0.01; one-way ANOVA; FIGS. 8A-8B). These experiments indicate thatCNO application significantly increases the firing of DREADDs-expressingPVN neurons.

Example 10: The Effects of CIH/H on OXT Receptor Activation

In order to examine if the release of OXT from PVN fibers is alteredwith CIH/H, the responses in OXT-sensitive CHO cells were examined inbrainstem tissue from unexposed sham and CIH/H exposed animals.Photoactivation of ChR2-containing PVN fibers in the DMV of brainstemslices from unexposed animals evoked large, reproducible, and transientincreases in intracellular calcium levels in OXT-sensitive CHO cells(average increases of 21.1±0.02% from baseline during first second;n=14; *p<0.05; one-way ANOVA with repeated measures; FIG. 9B, “shamanimals”). CHO cell responses upon PVN fiber stimulation in brainstemslices from animals exposed to CIH/H was significantly depressed(average increases of 8.1±0.01% from baseline during first second; n=16;*p<0.05; one-way ANOVA with repeated measures; FIG. 9B, “CIH/H animals”)compared to responses in unexposed animals (⁺p<0.05; one-way ANOVA; FIG.9B, “CIH/H animals”). These results indicate the release of OXT from PVNfibers in the DMV is significantly decreased following CIH/H exposure.To examine if restoration of OXT neuron function during CIH/H couldrestore responses in OXT-sensitive CHO cells, PVN OXT neurons wereactivated daily before and during CIH/H by daily injections of CNO toactivate PVN OXT neurons via excitation of DREADDs receptors in PVN OXTneurons. In animals with chronic activation of OXT neurons the responsesin OXT-sensitive CHO cells upon photostimulation of ChR2-containing PVNfibers in the DMV were restored and not significantly different fromresponses in air exposed control animals (average increases of23.4±0.03% from baseline during first second; n=17; *p<0.05; one-wayANOVA with repeated measures; FIG. 9B,“CIH/H+OXT neuron activatedanimals”). These restored responses in DREADDs-expressing animals werehowever, significantly increased compared to CIH/H exposed animals(⁺p<0.05; one way ANOVA; FIG. 9B, “CIH/H+OXT neuron activated animals”).These data indicate that OXT released from PVN fibers in the DMV isdiminished with CIH/H, but that this release can be restored withchronic PVN OXT neuron activation.

Example 11: Acute PVN OXT Neuron Activation Decreases Resting BloodPressure and Heart Rate

CNO administration had no effect on BP represented as mean arterialpressure (MAP) and HR in sham animals without DREADDs expression (FIGS.10A and 10B). However, CNO administration that activates DREADDsreceptors in PVN OXT neurons decreased resting HR and BP throughout 21days of CIH/H exposure, with significant decreases in MAP (104±2.6 mmHgbefore CNO to 93±1.7 mmHg after CNO; n=8; ⁺p<0.0001; paired t test; FIG.10A) and HR (416±7.1 beats/min before CNO to 362±10.5 beats/min afterCNO; n=8; ⁺p<0.01; paired t test; FIG. 10B). These data indicate thatselective activation of PVN OXT neurons decreases resting BP and HR inconscious unrestrained telemetry instrumented animals.

Example 12: Activation of Oxytocin Neurons Blunts the Increase in BPthat Occur with to Hypoxia/Hypercapnia

Activation of oxytocin neurons also blunted the increase in bloodpressure that occurred with hypoxia/hypercapnia (FIG. 11). Activation ofoxytocin neurons was achieved by selective expression and subsequentactivation of the excitatory Designer Receptors Exclusively Activated byDesigner Drugs (DREADDs) virally expressed in paraventricularhypothalamus oxytocin neurons upon microinjection of both anadeno-associated (AAV) floxed DREADDS virus and a lentivirus virus thatselectively drives CRE expression under the control of the oxytocinpromoter.

Example 13: Chronic PVN OXT Neuron Activation Prevents the Developmentof Hypertension that Occurs with CIH/H

To test if activation of PVN OXT neurons alters the changes in BP thatoccur with CIH/H, MAP was examined before and throughout 21 days ofCIH/H exposure in sham and OXT neuron activated animals. After 3 weeksof CIH/H, MAP increased to hypertensive levels in sham animals (from aMAP of 102±3.3 mmHg on control days to 120±0.5 mmHg on day 21; n=7;⁺p<0.01; one-way ANOVA with repeated measures; FIG. 12). Interestingly,animals receiving daily PVN OXT neuron activation experienced nosignificant changes in MAP throughout the 21 days of CIH/H exposure(from a MAP of 104±2.6 mmHg on control days to 103±3.0 mmHg on day 21;n=8; p>0.05; one-way ANOVA with repeated measures; FIG. 12). Animals inthe sham group experienced significant increases in MAP from day 12through day 21, whereas the increase in MAP was prevented in theDREADDs-expressing animals (Day 12: MAP in sham group of 117±4.1 mmHg,MAP in DREADDs-expressing group of 105±2.6 mmHg; Day 15: MAP in shamgroup of 118±2.6 mmHg, MAP in DREADDs-expressing group of 105±3.1 mmHg;Day 18: MAP in sham group of 122±1.1 mmHg, MAP in DREADDs-expressinggroup of 104±2.9 mmHg; Day 21: MAP in sham group of 120±0.5 mmHg, MAP inDREADDs-expressing group of 103±3.0 mmHg; n=7 sham animals and 8DREADDs-expressing animals; *p<0.01; two-way ANOVA with repeatedmeasures; FIG. 12). These data indicate that chronic activation of PVNOXT neurons prevents the development of hypertension that occurs in shamanimals with CIH/H exposure.

Example 14: Administration of Oxytocin to Human Patients Improves SleepQuality and Shows Cardio-Protective Effect

Patients recently diagnosed with OSA were recruited to test the effectof oxytocin administration on heart rate, apnea-hypopnea index, oxygensaturation, apnea duration, arousal index etc. The study is approved bythe FDA (IND #120989). Patients were administered 40 IU of oxytocinintranasally about one hour prior to sleeping.

The following eight outcomes were compared in the same patients prior toand after administration with oxytocin:

-   -   basal heart rate before sleep (primary outcome)    -   mean changes in heart rate with apneic and hypopneic events        (primary outcome)    -   apnea-hypopnea index (secondary outcome)    -   percentage of time spent by the patient with oxygen        saturations: >90%, >80% but <90%, and <80% (secondary outcome)    -   duration of apneas    -   oxygen desaturation during apneas (expressed as adverse        desaturations in percent oxygen)    -   numbers of arousals (expressed per hour)    -   sleep quality

The following information was recorded for each patient before and afterthe use of intranasal oxytocin:

-   1. Demographics—Age, gender, ethnicity, weight, BMI.-   2. Physiological data:    -   a. Basal heart rate before sleep    -   b. Apnea-hypopnea index    -   c. Mean changes in heart rate with apneic and hypopneic events    -   d. Percentage of time spent by the patient with oxygen        saturations: >90%, >80% but <90%, and <80%.    -   e. Duration of apneas    -   f. Oxygen desaturation during apneas (expressed as adverse        desaturations in percent oxygen)    -   g. Numbers of arousals (expressed per hour)    -   h. Sleep quality-   3. Standard of care sleep study data using PSG: Polysomnography    (PSG) monitors many body functions during sleep, including brain    (EEG), eye movements (EOG), muscle activity or skeletal muscle    activation (EMG), heart rhythm (ECG), respiratory airflow, thoracic    and abdominal respiratory effort, body position, limb movement, and    oxygen saturation using pulse oximetry. Recording and scoring was    done according to the standards set by the American Academy of Sleep    Medicine.-   4. Sleep quality score: Patients were asked to rank their responses    using the scale of 1-5 for a set of empirical factors:    -   1—Strongly disagree    -   2—Slightly disagree    -   3—Neither agree nor disagree    -   4—Slightly agree    -   5—Strongly agree

Empirical Factors:

-   -   I feel more refreshed than usual this morning    -   My quality of sleep last night was better than usual    -   I slept deeper than usual last night    -   I woke up fewer times than usual last night    -   I slept longer than usual last night    -   I feel better overall than usual this morning

The study shows that nasal administration of oxytocin reduces theduration of apnea (FIG. 13), reduces oxygen desaturation (FIG. 14),decreases the number of arousals/hour (FIG. 15), and improves sleepsatisfaction (FIG. 16).

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

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1-29. (canceled)
 30. A method for treating a patient suffering fromobstructive sleep apnea induced bradycardia, comprising: intranasallyadministering to the patient at least 40 International Units (IU) ofoxytocin within an hour of the patient falling asleep.
 31. The method ofclaim 30, wherein the administration of oxytocin reduces the incidenceof bradycardia associated with obstructive events.
 32. The method ofclaim 30, wherein the administration of oxytocin reduces the percentincidence of bradycardia associated with obstructive events by at least5%.
 33. The method of claim 30, wherein the administration of oxytocinincreases the respiratory rate in the patient during non-obstructiveperiods while sleeping.
 34. The method of claim 30, wherein theadministration of oxytocin increases the respiratory rate in the patientduring non-obstructive periods within 2 hours of sleep onset.
 35. Themethod of claim 30, wherein the administration of oxytocin increases therespiratory rate in the patient during non-obstructive periods whilesleeping by at least 0.5 breaths/min.
 36. The method of claim 30,wherein the patient is receiving continuous positive airway pressure(CPAP) therapy.
 37. The method of claim 30, wherein the administrationof oxytocin reduces the hypertension experienced by the patient.
 38. Themethod of claim 30, wherein the administration of oxytocin maintains theheart rate and/or blood pressure of the patient.
 39. The method of claim30, wherein the administration of oxytocin improves sleep quality in thepatient.
 40. The method of claim 30, wherein the administration ofoxytocin decreases the number of arousals per hour experienced by thepatient during sleep.
 41. The method of claim 30, wherein theadministration of oxytocin decreases the number of arousals per hourexperienced by the patient during sleep by at least 10%.
 42. The methodof claim 30, wherein the administration of oxytocin decreases the oxygendesaturation experienced by the patient during sleep.
 43. The method ofclaim 30, wherein the administration of oxytocin increases the minimumpercent oxygen saturation experienced by the patient during sleep by atleast 1%.
 44. The method of claim 30, wherein the administration ofoxytocin decreases the duration of apnea experienced by the patientduring sleep.
 45. The method of claim 30, wherein the administration ofoxytocin decreases the duration of apnea experienced by the patientduring sleep by at least 5%.
 46. The method of claim 30, wherein theadministration of oxytocin decreases the duration of apnea experiencedby the patient during sleep by at least 10%.
 47. A method for increasingnocturnal respiration rate in a patient in need thereof, comprising:intranasally administering to the patient at least 40 InternationalUnits (IU) of oxytocin within an hour of the patient falling asleep. 48.The method of claim 47, wherein the administration of oxytocin increasesthe respiratory rate of the patient during non-obstructive periods whilesleeping.
 49. The method of claim 47, wherein the administration ofoxytocin increases the respiratory rate of the patient duringnon-obstructive periods within 2 hours of sleep onset.
 50. The method ofclaim 47, wherein the administration of oxytocin increases therespiratory rate in the patient during non-obstructive periods whilesleeping by at least 0.5 breaths/min.
 51. The method of claim 47,wherein the administration of oxytocin increases the respiratory rate inthe patient during non-obstructive periods while sleeping by at least0.5 breaths/min within 2 hours of sleep onset.
 52. A kit for improvingsleep quality in a patient suffering from obstructive sleep apnea,comprising: a dose of at least 40 International Units (IU) of oxytocin;means for intranasal administration of the oxytocin dose; andinstructions for use of the oxytocin dose less than one hour prior tosleep onset.
 53. A kit for treating obstructive sleep apnea in a patientreceiving continuous positive airway pressure (CPAP) therapy,comprising: a dose of at least 40 International Units (IU) of oxytocin;and means for administration of the oxytocin dose via the CPAP machinewithin an hour of sleep onset.
 54. A therapeutic device for treatingobstructive sleep apnea in a patient receiving continuous positiveairway pressure (CPAP) therapy, comprising: a CPAP machine; a dose of atleast 40 International Units (IU) of oxytocin; and means foradministration of the oxytocin dose via the CPAP machine within an hourof sleep onset.
 55. A composition comprising a dose of at least 40International Units (IU) of oxytocin, wherein the composition iscontained in a device for intranasal administration, and wherein thedevice administers at least 40 IU of oxytocin at one time.